Fig. 6: APT2 modulates K48-linked polyubiquitination of TBK1 through TRIM27.

a HEK293T cells were transfected with FLAG-TBK1 and HA-tagged WT ubiquitin (HA-Ub) or its mutants, together with MYC-EV or MYC-APT2 for 24 h, followed by treatment with Baf A1 (0.2 μM) for 6 h. Cell lysates were collected for IP (with anti-FLAG) and immunoblot analysis. This was repeated n = 3 independent times with similar results. b HEK293T cells were transfected with FLAG-TBK1 and HA-K48-linked ubiquitin for 24 h, followed by treatment with ML349 at indicated concentrations for 12 h. Cell lysates were collected for IP (with anti-FLAG) and immunoblot analysis. This was repeated n = 3 independent times with similar results. c WT or Lypla2-KO RAW 264.7 cells were stimulated with gDNA for 6 h, followed by treatment with Baf A1 (0.2 μM) for 6 h. Cell lysates were collected for IP (with anti-TBK1) and immunoblot analysis. This was repeated n = 3 independent times with similar results. d, e WT (d) and TBK1-KO (e) HEK293T cells were transfected with the indicated plasmids, followed by treatment with Baf A1 (0.2 μM) for 6 h. Cell lysates were collected for IP (with anti-FLAG) and immunoblot analysis. This was repeated n = 3 independent times with similar results. f HEK293T cells were transfected with Scr shRNA or other E3 ligase-specific shRNAs for 24 h, followed by transfected with FLAG-TBK1, together with HA-K48-linked ubiquitin and MYC-EV or MYC-APT2 for 24 h, and then followed by treatment with Baf A1 (0.2 μM) for 6 h. Cell lysates were collected for IP (with anti-FLAG) and immunoblot analysis. This was repeated n = 3 independent times with similar results. g, h HEK293T cells were transfected with FLAG-EV, WT FLAG-APT2 or its catalytically inactivated variants for 24 h (g). TBK1-KO HEK293T cells were reconstituted with plasmids encoding WT FLAG-TBK1 or FLAG-TBK1 C292S mutant for 24 h (h). The colocalization between TBK1 and TRIM27 was examined by PLA. The nucleus was stained with DAPI. Scale, 10 μm. Quantitative analyses of the colocalization (10 cells per group) is next to the image. This was repeated n = 3 independent times with similar results. i PEMs were stimulated with freeze-thawed iRBCs at the indicated time points. Cell lysates were collected for IP and immunoblot analysis. This was repeated n = 3 independent times with similar results. j PEMs were stimulated with freeze-thawed iRBCs for 12 h. The colocalization between APT2 (red) and TRIM27 (green) was examined by confocal microscopy. The nucleus was stained with DAPI. Scale, 10 μm. The intensity analysis is next to the image. This was repeated n = 3 independent times with similar results. k HEK293T cells were transfected with WT FLAG-TBK1 or its K251R/K372R (2KR) mutant, together with HA-K48-linked ubiquitin and MYC-EV or MYC-APT2 for 24 h, followed by treatment with Baf A1 (0.2 μM) for 6 h. Cell lysates were collected for IP (with anti-FLAG) and immunoblot analysis. This was repeated n = 3 independent times with similar results. l HEK293T cells were transfected with WT FLAG-TBK1 or its 2KR mutant, together with MYC-EV or MYC-APT2 for 24 h. Cell lysates were collected for immunoblot analysis. This was repeated n = 3 independent times with similar results. m HEK293T cells were transfected with WT FLAG-TBK1 or its 2KR mutant, together with a luciferase reporter for IFN-β-luc and MYC-EV or MYC-APT2 for 24 h. Cell lysates were collected for luciferase reporter assays (n = 3 biological replicates). Data are presented as the mean ± SD. P-values were determined by unpaired two tailed Student’s t tests (g, h, m). Source data are provided as a Source Data file.